Protein-polymer conjugates

ABSTRACT

This invention relates to protein-polymer conjugates described in the specification. Also disclosed are a method for preparing a protein-polymer conjugate and its use in treating hepatitis B virus or hepatitis C virus infection.

CROSS-REFERENCE TO RELATED APPLICATION PARAGRAPH

This application claims the benefit of U.S. Provisional Application No.60/956,273 filed on Aug. 16, 2007. The contents of which, is herebyincorporated by reference in its entirety

BACKGROUND

Advance in cell biology and recombinant protein technologies has led tothe development of protein therapeutics.

Yet, major hurdles still exist. Most proteins are susceptible toproteolytic degradation and therefore have a short half-life in thecirculating system. Other disadvantages include low water solubility andinducement of neutralizing antibodies.

Attachment of a polymer, e.g., polyethylene glycol (PEG), to a proteinhinders access of proteolytic enzymes to the protein backbone, resultingin enhanced protein stability. In addition, it may also improve watersolubility and minimize immunogenicity. There is a need for effectivemethods of attaching polymer to proteins.

SUMMARY

An aspect of the present invention relates to a substantially pure (≧80%pure) conjugate containing one or more polymer moieties, a proteinmoiety, and a linker. In the conjugate, the polymer moiety or moietiesare attached to the linker; the nitrogen atom of the N-terminus of theprotein moiety is bonded to the linker; the linker is a covalent bond,C₁₋₁₀ alkylene, C₂₋₁₀ alkenylene, or C₂₋₁₀ alkynylene; and the proteinmoiety is an interferon-α moiety, a human growth hormone moiety, or anerythropoietin moiety. Preferably, the conjugate has a purity of 90% orhigher. This conjugate has an unexpected long in vivo half-life.

Another aspect of the present invention relates to protein-polymerconjugates of formula I:

in which each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C₁₋₅alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, aryl, heteroaryl, C₃₋₈ cycloalkyl, orC₃₋₈ heterocycloalkyl; each of A₁ and A₂, independently, is a polymermoiety; each of G₁, G₂, and G₃, independently, is a bond or a linkingfunctional group; P is a protein moiety; m is 0 or an integer of 1-10;and n is an integer of 1-10.

Referring to the above formula, the protein-polymer conjugate may haveone or more of the following features: G₃ is a bond and P is a proteinmoiety in which the amino group at the N-terminus is attached to G₃; A₁and A₂ are polyalkylene oxide moieties having a molecular weight of2-100 kD (preferably 10-30 kD), each of G₁ and G₂ is

(in which O is attached to A₁ or A₂, and NH is attached to a carbon atomas shown in formula I), or each of G₁ and G₂ is urea, sulfonamide, oramide, (in which N is attached to a carbon atom as shown in formula I);m is 4, n is 2, and each of R₁, R₂, R₃, R₄, and R₅ is H; and P is aninterferon moiety or a modified interferon moiety containing 1-4additional amino acid residues.

The term “alkyl” refers to a mono-valent straight-chained or branchedhydrocarbon radical. Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, tert-butyl, and n-pentyl. Similarly, the term“alkenyl” or “alkynyl” refers to a mono-valent straight-chained orbranched hydrocarbon radical containing one or more C═C double bonds orone or more C≡C triple bonds.

The term “alkylene” refers to a bi-valent straight-chained or branchedhydrocarbon radical. Similarly, the term “alkenylene” or “alkynylene”refers to a bi-valent straight-chained or branched hydrocarbon radicalcontaining one or more C═C double bonds or one or more C≡C triple bonds.

The term “aryl” refers to a hydrocarbon ring system (mono-cyclic orbi-cyclic) having at least one aromatic ring. Examples of aryl moietiesinclude, but are not limited to, phenyl, naphthyl, and pyrenyl.

The term “heteroaryl” refers to a hydrocarbon ring system (mono-cyclicor bi-cyclic) having at least one aromatic ring which contains at leastone heteroatom such as O, N, or S as part of the ring system and thereminder being carbon. Examples of heteroaryl moieties include, but arenot limited to, furyl, pyrrolyl, thienyl, oxazolyl, imidazolyl,thiazolyl, pyridinyl, pyrimidinyl, quinazolinyl, and indolyl.

The term “cycloalkyl” refers to a partially or fully saturatedmono-cyclic or bi-cyclic ring system having only carbon ring atoms.Examples include, but are not limited to, cyclopropanyl, cyclopentanyl,and cyclohexanyl.

The term “heterocycloalkyl” refers to a partially or fully saturatedmono-cyclic or bi-cyclic ring system having, in addition to carbon, oneor more heteroatoms (e.g., O, N, or S), as ring atoms. Examples include,but are not limited to, piperidine, piperazine, morpholine,thiomorpholine, and 1,4-oxazepane.

Alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, andheterocycloalkyl mentioned herein include both substituted andunsubstituted moieties. Examples of substituents include C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₈ cycloalkyl, C₅-C₈ cycloalkenyl,C₁-C₁₀ alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C₁-C₁₀alkylamino, C₁-C₂₀ dialkylamino, arylamino, diarylamino, hydroxyamino,alkoxyamino, C₁-C₁₀ alkylsulfonamide, arylsulfonamide, hydroxy, halogen,thio, C₁-C₁₀ alkylthio, arylthio, cyano, nitro, acyl, acyloxy, carboxyl,and carboxylic ester.

The term “polyalkylene oxide moiety” refers to a mono-valent radicalderived from linear, branched, or star-shaped polyalkylene oxide. Themolecular weight of a polyalkylene oxide moiety may be 2-100 kD. Thepolyalkylene oxide moiety is either saturated or unsaturated. Examplesof a polyalkylene oxide moiety include, but are not limited to,polyethylene oxide, polyethylene glycol, polyisopropylene oxide,polybutenylene oxide, and copolymers thereof. Other polymers such asdextran, polyvinyl alcohols, polyacrylamides, or carbohydrate-basedpolymers can also be used to replace the polyalkylene oxide moiety, aslong as they are not antigenic, toxic, or eliciting immune response. Thepolyalkylene oxide moiety is either substituted or unsubstituted. Forexample, it can be methoxy-capped polyethylene glycol (mPEG).

The term “protein moiety” refers to a mono-valent radical derived fromeither a naturally occurring protein or a modified protein. Thenaturally occurring protein can be interferon-α, interferon-β, humangrowth hormone, erythropoietin and granulocyte colon-stimulating factor,or antibody. The modified protein can be, e.g., a protein containinginterferon-α and 1-4 additional amino acid residues at the N-terminus ofthe interferon. An example of such a modified interferon is

IFN representing an interferon-α_(2b) moiety, the amino group at theN-terminus of which is bonded to the carbonyl group.

The term “interferon-α” refers to a family of highly homologousspecies-specific proteins that inhibit viral replication and cellularproliferation and modulate immune response. See Bonnem et al., J. Biol.Response Mod., 1984, 3(6):580-598; and Finter, J. Hepatol., 1986, 3Suppl 2:S157-160.

Many types of interferon-α proteins are commercially available,including Intron-A interferon provided by Schering Corporation,Kenilworth, N.J., Roferon interferon provided by Hoffmann-La Roche,Nutley, N.J., Berofor alpha 2 interferon provided by BoehringerIngelheim Pharmaceutical, Inc., Ridgefield, Conn., Sumiferon provided bySumitomo, Japan, and Wellferon interferon alpha-n1 (INS) provided byGlaxo-Wellcome Ltd., London, Great Britain.

Listed below are amino acid sequences of five exemplary humaninterferon-α proteins, either in precursor form or in mature form:

maltfallva llvlsckssc svgcdlpqth slgsrrtlml laqmrrislf sclkdrhdfgfpqeefgnqf qkaetipvlh emiqqifnlf stkdssaawd etlldkfyte lyqqlndleacviqgvgvte tplmkedsil avrkyfqrit lylkekkysp cawevvraei mrsfslstnlqeslrske

(See Krasagakis et al., Cancer Invest. 26 (6), 562-568, 2008)

cdlpqthslg srrtlmllaq mrkislfscl kdrhdfgfpq eefgnqfqka etipvlhemiqqifnlfstk dssaawdetl ldkfytelyq qlndleacvi qgvgvtetpl mkedsilavrkyfqritlyl kekkyspcaw evvraeimrs fslstnlqes lrske

(See Klaus, et al., J. Mol. Biol. 274 (4), 661-675, 1997)

mcdlpqthsl gsrrtlmlla qmrrislfsc lkdrhdfgfp qeefgnqfqk aetipvlhemiqqifnlfst kdssaawdet lldkfytely qqlndleacv iqgvgvtetp lmkedsilavrkyfqritly lkekkyspca wevvraeimr sfslstnlqe slrske

(See GenBank Accession Number AAP20099, the 30-APR-2003 version.)

mallfpllaa lvmtsyspvg slgcdlpqnh gllsrntlvl lhqmrrispf lclkdrrdfrfpqemvkgsq lqkahvmsvl hemlqqifsl fhterssaaw nmtlldqlht elhqqlqhletcllqvvgeg esagaisspa ltlrryfqgi rvylkekkys dcawevvrme imkslflstnmqerlrskdr dlgss

(See Capon et al., J. Mol. Cell. Biol. 5 (4):768-779, 1985)

lsyksicslg cdlpqthslg nrralillaq mgrispfscl kdrhdfglpq eefdgnqfqktqaisvlhem iqqtfnlfst edssaaweqs llekfstely qqlnnleacv iqevgmeetplmnedsilav rkyfqritly ltekkyspca wevvraeimr slsfstnlqk rlrrkd

(See Lund et al., J. Interferon Res. 5 (2), 229-238, 1985)

In one example, the interferon-α protein used for making the conjugateof this invention has an amino acid sequence at least 80% (e.g., 85%,90%, 95%, or 99%) identical to one of the above listed amino acidsequences, or to the fragment thereof that corresponds to a matureinterferon alpha.

The term “human growth hormone” refers to the naturally occurring humangrowth hormone, either in precursor or mature form, and its functionalvariants, i.e., having an amino acid sequence at least 80% (e.g., 85%,90%, 95%, or 99%) identical to the naturally occurring human growthhormone and possessing the same physiological activity of that humangrowth hormone. The amino acid sequences of the naturally occurringhuman growth hormone (in precursor and mature form) are shown below:

matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla fdtyqefeeayipkeqkysf lqnpqtslcf sesiptpsnr eetqqksnle llrisllliq swlepvqflrsvfanslvyg asdsnvydll kdleegiqtl mgrledgspr tgqifkqtys kfdtnshnddallknyglly cfrkdmdkve tflrivqcrs vegscgf (precursor) fptiplsrlfdnamlrahrl hqlafdtyqe feeayipkeq kysflqnpqt slcfsesipt psnreetqqksnlellrisl lliqswlepv qflrsvfans lvygasdsnv ydllkdleeg iqtlmgrledgsprtgqifk qtyskfdtns hnddallkny gllycfrkdm dkvetflriv qcrsvegscg f(mature form)

Erythropoietin (EPO), produced by either liver or kidney, is aglycoprotein hormone that controls erythropoiesis or red blood cellproduction. See U.S. Pat. No. 5,621,080. The amino acid sequences ofhuman EPO (in precursor and mature form) are shown below:

mgvhecpawl wlllsllslp lglpvlgapp rlicdsrvle rylleakeae nittgcaehcslnenitvpd tkvnfyawkr mevgqqavev wqglallsea vlrgqallvn ssqpweplqlhvdkavsglr slttllralg aqkeaisppd aasaaplrti tadtfrklfr vysnflrgklklytgeacrt gdr (precursor) apprlicdsr vlerylleak eaekittgca ehcslnekitvpdtkvnfya wkrmevgqqa vevwqglall seavlrgqal lvkssqpwep lqlhvdkavsglrslttllr algaqkeais ppdaasaapl rtitadtfrk lfrvysnflr gklklytgea crtgdr(mature form)

An erythropoietin protein used to make the conjugate of this inventioncan be an EPO protein, either in precursor or mature form, produced by asuitable species, e.g., human, murine, swine, and bovine. In oneexample, the erythropoietin protein has an amino acid sequence at least80% (e.g., 85%, 90%, 95% or 99%) identical to one of the amino acidsequences shown above.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of the invention. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

The term “linking functional group” refers to a bi-valent functionalgroup, one end being connected to the polymer moiety and the other endbeing connected to the protein moiety. Examples include, but are notlimited to, —O—, —S—, carboxylic ester, carbonyl, carbonate, amide,carbamate, urea, sulfonyl, sulfinyl, amino, imino, hydroxyamino,phosphonate, or phosphate group.

The protein-polymer conjugate described above can be in the free form orin the form of salt, if applicable. A salt, for example, can be formedbetween an anion and a positively charged group (e.g., amino) on aprotein-polymer conjugate of this invention. Suitable anions includechloride, bromide, iodide, sulfate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt canalso be formed between a cation and a negatively charged group (e.g.,carboxylate) on a protein-polymer conjugate of this invention. Suitablecations include sodium ion, potassium ion, magnesium ion, calcium ion,and an ammonium cation such as tetramethylammonium ion.

In addition, the protein-polymer conjugate may have one or more doublebonds, or one or more asymmetric centers. Such a conjugate can occur asracemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double bond isomeric forms.

An example the protein-polymer conjugate of this invention is shownbelow:

in which mPEG has a molecular weight of 20 kD and IFN is aninterferon-α_(2b) moiety.

Still another aspect of this invention relates to compounds which areuseful for preparing protein-polymer conjugates. The compounds haveformula TI:

in which X is a reactive group; each of A₁ and A₂, independently, is apolymer moiety, or one of A₁ and A₂ is a polymer moiety, and the otheris a reactive group; each of G₁, G₂, and G₃, independently, is a bond ora linking functional group; each of R₁, R₂, R₃, R₄, and R₅,independently, is H, C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, aryl,heteroaryl, C₃₋₈ cycloalkyl, C₃₋₈ heterocycloalkyl, or -G₄-X′, G₄ beinga bond or a linking functional group and X′ being a reactive group; eachm and n, independently, is 0 or an integer of 1-10; provided that if-G₃-X is —CO(O)—N-succinimidyl, then n is an integer of 1-10.

In one embodiment, X, X′, A₁, A₂, G₁, G₂, G₃, G₄, R₁, R₂, R₃, R₄, R₅,and m are defined above, and n is an integer of 1-10.

Certain compounds of formula II have one or more of the followingfeatures: the polymer moiety may have one or more reactive groups; G₃ isa bond and P is a protein moiety in which the amino group at theN-terminus is attached to G₃; A₁ and A₂ are polyalkylene oxide moietieshaving a molecular weight of 2-100 kD (preferably 12-30 kD), each of G₁and G₂ is

in which O is attached to A₁ or A₂, and NH is attached to a carbon atomas shown in formula I; m is 4; n is 1; each of R₁, R₂, R₃, R₄, and R₅ isH; and X is CH(═O) or X is a leaving group (e.g., succinimidyl orp-nitrophenoxy).

The term “reactive group” refers to a functional group that can reactwith another functional group so that it is either replaced by the otherfunctional group, or is linked to the other function group. A reactivegroup can be a leaving group, a nucleophilic group, an aldehyde, or aMichael receptor.

The term “leaving group” refers to a functional group that can depart,upon direct displacement or ionization, with the pair of electrons fromone of its covalent bonds (see, e.g., F. A. Carey and R. J. Sunberg,Advanced Organic Chemistry, 3rd Ed. Plenum Press, 1990). Examples of aleaving group include, but are not limited to, methanesulfonate,triflate, p-toluenesulfonate, iodine, bromide, chloride,trifluoroacetate, succinimidyl (“Su”), p-nitrophenoxy, andpyridine-2-yl-oxy.

The term “nucleophilic group” refers to an electron-rich functionalgroup, which reacts with an electron-receiving group, such aselectrophile, by donating an electron pair.

The term “electrophilic group” refers to an electron-poor functionalgroup, which reacts with an electron-donating group, such as anucleophile, by accepting an electron pair. Michael receptors are asubset of electrophilic groups. They, upon contacting a nucleophile,undergo Michael reaction. A typical Michael receptor contains anα,β-unsaturated ketone moiety.

Exemplary compounds of formula II are shown below:

In still another aspect, this invention features a method of preparingprotein-polymer conjugates of formula I, in which the amino group at theN-terminus of protein moiety P is attached to linking functional groupG₃. The method includes coupling an N-terminus free protein H—P with adi-polymer branched molecule of a formula the same as formula II shownabove, except that X is a leaving group. The N-terminus free protein H—Prefers to a protein in which the nitrogen atom at the terminal aminogroup of the protein moiety P is bonded to the hydrogen atom H. In otherwords, H—P has a terminal primary or secondary amino group.Alternatively, the method includes (1) coupling the just-mentionedN-terminus free protein H—P with a di-polymer branched molecule of aformula the same as formula II shown above, except that G₃ is a bond, nis 0 or an integer of 1-9, and X is CHO; and (2) reducing the couplingproduct to form a protein-polymer conjugate.

Interferon is an immunomodulating medication for treating hepatitis Bvirus (HBV) infection or hepatitis C virus (HCV) infection. In addition,interferon (e.g., interferon-α) can be used to treat non-Hodgkin'slymphoma, Hairy cell leukemia, chronic myelogenous leukemia,AIDS-related Kaposis sarcoma, follicular lymphoma, malignant melanoma,and condyloma accuminata. Thus, another aspect of this invention relatesto a method of treating any of the above-mentioned disorders by aninterferon-polymer conjugate described herein.

Also within the scope of this invention is a composition containing thean interferon-polymer conjugate for use in any of the above-mentioneddisorders, as well as this therapeutic use and use of the conjugate forthe manufacture of a medicament for treating one of these disorders.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DETAILED DESCRIPTION

Protein-polymer conjugates of the present invention can be prepared bysynthetic methods well known in the chemical art. For example, one canbond a linker molecule, concurrently or separately, with two polymermolecules and subsequently bond a protein molecule to the linkermolecule to form a protein-polymer conjugate of this invention.

Also within the scope of this invention are the compounds of formula(II). These compounds are useful for making the just-describedprotein-polymer conjugates. They can be prepared by synthetic methodswell known in the chemical art. An illustrative synthetic scheme and anactual example are provided below.

Scheme 1 shows an example of preparing protein-polymer conjugates ofthis invention. Diamine compound 1, which contains an acetal group, isreacted with N-hydroxysuccinimidyl carbonate mPEG (i.e., compound 2) toform di-PEGylated compound 3, which is subsequently converted toaldehyde 4. This aldehyde compound is reacted with protein having a freeamino group via reductive alkylation to afford a protein-polymerconjugate of this invention.

A protein-polymer conjugate thus synthesized can be further purified bya method such as ion exchange chromatography, gel filtrationchromatography, electrophoresis, dialysis, ultrafiltration, orultracentrifugation.

The chemical reactions described above include using solvents, reagents,catalysts, protecting group and deprotecting group reagents, and certainreaction conditions. They may additionally include steps, either beforeor after the steps described specifically herein, to add or removesuitable protecting groups in order to ultimately allow for synthesis ofa protein-polymer conjugate. In addition, various synthetic steps may beperformed in an alternate sequence or order to give the desiredprotein-polymer conjugates. Synthetic chemistry transformations andprotecting group methodologies (protection and deprotection) useful insynthesizing applicable protein-polymer conjugates are known in the artand include, for example, those described in R. Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley andSons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995) and subsequent editions thereof.

The conjugate of the invention has a very high purity. Namely, in 80% ormore of the conjugate molecules, the polymer moiety is linked to thesame nitrogen atom of the N-terminus of the same protein moiety. Inother words, in at least 80% of the conjugate molecules, the proteinmoiety is identical in all aspects, including its sequence and itsbonding position to the polymer moiety.

The conjugate of the invention may be pharmaceutically active in theconjugate form. Alternatively, it can release a pharmaceutically activeprotein in vivo (e.g., through hydrolysis) by enzymatically cleaving thelinkage between the protein moiety and the polymer moiety. Examples ofenzymes involved in in vivo cleaving linkages include oxidative enzymes(e.g., peroxidases, amine oxidases, or dehydrogenases), reductiveenzymes (e.g., keto reductases), and hydrolytic enzymes (e.g.,proteases, esterases, sulfatases, or phosphatases).

Thus, one aspect of this invention relates to a method of administeringan effective amount of one or more of the above-describedprotein-polymer conjugates for treating a disease (e.g., HBV or HCVinfection). Specifically, a disease can be treated by administering to asubject one or more of the protein-polymer conjugates in an effectiveamount. Such a subject can be identified by a health care professionalbased on results from any suitable diagnostic method.

As used herein, the term “treating” or “treatment” is defined as theapplication or administration of a composition including aprotein-polymer conjugate to a subject (human or animal), who has adisorder, a symptom of the disorder, a disease or disorder secondary tothe disorder, or a predisposition toward the disorder, with the purposeto cure, alleviate, relieve, remedy, or ameliorate the disorder, thesymptom of the disorder, the disease or disorder secondary to thedisorder, or the predisposition toward the disorder. “An effectiveamount” refers to an amount of a protein-polymer conjugate which confersa therapeutic effect on the treated subject. The therapeutic effect maybe objective (i.e., measurably by some tests or markers) or subjective(i.e., a subject gives an indication of or feels an effect).

Also within the scope of this invention is a pharmaceutical compositioncontains an effective amount of at least one of the protein-polymerconjugates described above and a pharmaceutical acceptable carrier.Further, this invention includes a method of administering an effectiveamount of one or more of the protein-polymer conjugates to a patientwith one or more diseases. Effective doses will vary, as recognized bythose skilled in the art, depending on, e.g., the rate of hydrolysis ofa protein-polymer conjugate, the types of diseases to be treated, theroute of administration, the excipient usage, and the possibility ofco-usage with other therapeutic treatment.

To practice the method of the present invention, a composition havingone or more of the above-mentioned compounds can be administeredparenterally, orally, nasally, rectally, topically, or buccally. Theterm “parenteral” as used herein refers to subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional,intraperitoneal, intratracheal or intracranial injection, as well as anysuitable infusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent, such as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution, and isotonic sodiumchloride solution. In addition, fixed oils are conventionally employedas a solvent or suspending medium (e.g., synthetic mono- ordi-glycerides). Fatty acid, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents. Other commonlyused surfactants such as Tweens or Spans or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms can also be used for the purpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions, and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.

A nasal aerosol or inhalation composition can be prepared according totechniques well known in the art of pharmaceutical formulation. Forexample, such a composition can be prepared as a solution in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art. A composition havingone or more of the above-described compounds can also be administered inthe form of suppositories for rectal administration.

A pharmaceutically acceptable carrier is routinely used with one or moreactive above-mentioned compounds. The carrier in the pharmaceuticalcomposition must be “acceptable” in the sense that it is compatible withthe active ingredient of the composition (and preferably, capable ofstabilizing the active ingredient) and not deleterious to the subject tobe treated. One or more solubilizing agents can be utilized aspharmaceutical excipients for delivery of an above-mentioned compound.Examples of other carriers include colloidal silicon oxide, magnesiumstearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.

Suitable in vitro assays can be used to preliminarily evaluate theefficacy of the above-described conjugates in inhibiting hepatitis Bvirus or hepatitis C virus. The conjugates can further be examined forits efficacy in treating hepatitis B virus or hepatitis C virusinfection by in vivo assays. For example, they can be administered to ananimal (e.g., a mouse model) or a human having hepatitis B virus orhepatitis C virus infection and its therapeutic effects are thenassessed. Based on the results, dosage ranges and administration routescan also be determined.

The example below is to be construed as merely illustrative, and notlimitative of the remainder of the disclosure in any way whatsoever.Without further elaboration, it is believed that one skilled in the artcan, based on the description herein, utilize the present invention toits fullest extent. All publications cited herein are herebyincorporated by reference in their entirety.

Preparation of di-PEG Aldehyde

20 kD PEGO(C═O)OSu was prepared from 20 kD mPEGOH purchased from (SunBioInc., CA, USA) according to the method described in Bioconjugate Chem.1993, 4, 568-569.

A solution of 6-(1,3-dioxolan-2-yl)hexane-1,5-diamine in dichloromethane(11.97 g of the solution containing 9.03 mg of diamine, 47.8 μmol) wasadded to a flask containing 20 kD PEGO(C═O)OSu (1.72 g, 86.0 μmol).After PEGO(C═O)OSu was completely dissolved, N,N-diisopropylethylamine(79 μL, 478 μmol) was added. The reaction mixture was stirred at roomtemperature for 24 h, and then methyl t-butyl ether (200 mL) was addeddropwise with stirring. The resulting precipitate was collected anddried under vacuum to give di-PEG acetal (1.69 g, 98%) as a white solid.

¹H NMR (400 MHz, d₆-DMSO) δ 7.16 (t, J=5.2 Hz, 1H), 7.06 (d, J=8.8 Hz,1H), 4.76 (t, J=4.8 Hz, 1H), 4.10-3.95 (m, 4H), 1.80-1.65 (m, 1H),1.65-1.50 (m, 1H), 1.48-1.10 (m, 6H).

Di-PEG acetal (4.0 g, 0.2 mmol) was suspended in pH 2.0 buffer (criticacid, 40 mL). The reaction mixture was stirred at 35° C. for 24 h andthen extracted with dichloromethane (3×50 mL). The combined organiclayers were dried over magnesium sulfate, concentrated, and thenre-dissolved in dichloromethane (20 mL). The solution was addeddropwisely to methyl t-butyl ether (400 mL) with stirring. The resultingprecipitate was collected and dried at reduced pressure to give di-PEGaldehyde (3.8 g, 95%) as a white solid. ¹H NMR (400 MHz, d₆-DMSO) δ 9.60(s, 1H), 7.24 (d, J=8.4 Hz, 1H), 7.16 (t, J=5.2 Hz, 1H), 4.10-3.95 (m,4H), 3.95-3.80 (m, 1H), 3.00-2.85 (m, 2H), 2.58-2.36 (m, 2H), 1.46-1.15(m, 6H).

Preparation of Modified Interferon

A modified recombinant human interferon-α_(2b) was cloned by a PCRmethod using human genomic DNA as a template. The oligonucleotides weresynthesized based on the flanking sequences of human interferon-α_(2b)(GenBank Accession #J00207, Jan. 8, 2008). The derived PCR products weresubcloned into pGEM-T vector (Promega). The IFN variant was PCRamplified again through the pGEM-T clones and subsequently subclonedinto protein expression vector pET-24a (Novagen), a T7 RNA polymerasepromoter driven vector, using NdeI/BamHI as the cloning sites. VectorpET-24a was then transformed into E. coli BL21-CodonPlus (DE 3)-RIL(Stratagene) strain. The high-expression clones were selected bymaintaining the transformed E. coli BL21-CodonPlus (DE 3)-RIL in thepresence of karamycin (50 μg/mL) and chloramphenical (50 μg/mL).

Terrific broth medium (BD, 200 mL) was employed for the propagation ofBL21-CodonPlus (DE 3)-RIL with Pro-IFN gene in a 1000 mL flask. Theflask was shaken at 37° C. at 230 rpm for 16 hr. Batch and fed-batchfermentations were performed in a 5-liter jar fermentor (Bioflo 3000;New Brunswick Scientific Co., Edison, N.J.). The batch fermentation used150 mL of an overnight preculture inoculum and 3 L of the Terrific brothmedium with karamycin (50 μg/mL), chloramphenical (50 ug/mL), 0.4%glycerol, and 0.5% (v/v) trace elements (10 g/L of FeSO₄·7H₂O, 2.25 g/Lof ZnSO₄·7H₂O, 1 g/L of CuSO₄·5H₂O, 0.5 g/L of MnSO₄H₂O, 0.3 g/L ofH₃BO₃, 2 g/L of CaCl₂·2H₂O, 0.1 g/L of (NH₄)₆Mo₇O₂₄, 0.84 g L EDTA, 50ml/L HCl). The dissolved oxygen concentration was controlled at 35% andthe pH was kept at 7.2 by adding a 5 N NaOH aqueous solution. A feedingsolution containing 600 g/L of glucose and 20 g/L of MgSO₄·7H₂O wasprepared. When the pH rose to a value greater than the set point, anappropriate volume of the feeding solution was added to increase theglucose concentration in the culture broth. Expression of the Pro-IFNgene was induced by adding IPTG to a final concentration of 1 mM and theculture broth was harvested after incubating for 3 hr.

The collected cell pellet was resuspended with TEN buffer (50 mMTris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl) in an approximate ratio of1:10 (wet weight g/mL) and disrupted by a microfluidizer, and thencentrifuged at 10,000 rpm for 20 min. The pellet containing inclusionbody (IB) was washed twice with TEN buffer and centrifuged as describedabove. The pellet containing IB was then suspended in 150 mL of a 4 Mguanidium HCl (GuHCl) aqueous solution and centrifuged at 20,000 rpm for15 min. The IB was then solubilized in 50 mL of 6 M GuHCl solution. TheGuHCl solubilized material was centrifuged at 20,000 rpm for 20 min.Refolding was initiated by dilution of denatured IB in 1.5 L of afreshly prepared refolding buffer (100 mM Tris-HCl (pH 8.0), 0.5 ML-Arginine, 2 mM EDTA) that was stirred only during the addition. Therefolding reaction mixture was allowed to incubate for 48 hr withoutstirring. The refolded recombinant human interferon-α_(2b) (i.e.,Pro-IFN) was dialyzed against 20 mM Tris buffer (with 2 mM EDTA and 0.1Murea, pH 7.0) for further purification by Q-Sepharose columnchromatography.

The refolded recombinant human protein Pro-IFN was loaded onto aQ-Sepharose column (GE Amersham Pharmacia, Pittsburgh, Pa.). The columnwas pre-equilibrated and washed with a 20 mM Tris-HCl buffer (pH 7.0).The product was eluted with a mixture of 20 mM Tris-HCl buffer (pH 7.0)and 200 mM NaCl. Fractions containing Pro-IFN was collected based on itsabsorbance at 280 nm. The concentration of Pro-IFN was determined by aprotein assay kit using the Bradford method (Pierce, Rockford, Ill.).

Prepare Protein-Polymer Conjugate

To a solution of di-PEG aldehyde prepared above (1.2 g, 0.03 mmol) inwater (72 mL) was added 2 M sodium phosphate buffer (pH 4.0, 5 mL) andPro-IFN (200 mg in 22.2 mL of pH 7.0 buffer containing 20 mM Tris-HCland 0.2M NaCl, 0.01 mmol). The reaction mixture was stirred at roomtemperature for 10 min; then sodium cyanoborohydride aqueous solution(400 mM, 1.25 mL, 0.5 mmol) was added. The reaction mixture was stirredin the dark for 16 h and purified by SP XL Sepharose chromatography.Fractions containing the desired polymer-protein conjugate werecollected based on their retention time and absorbance at 280 nm. Theconcentration of the conjugate was determined by a protein assay kitusing the Bradford method (Pierce, Rockford, Ill.). The isolated yieldof the conjugate was roughly 40% or higher.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of thefollowing claims.

1. A substantially pure conjugate comprising at least one polymermoiety, a protein moiety, and a linker, wherein the at least one polymermoiety is attached to the linker; the nitrogen atom of the N-terminus ofthe protein moiety is bonded to the linker; the linker is a covalentbond, C₁₋₁₀ alkylene, C₂₋₁₀ alkenylene, or C₂₋₁₀ alkynylene; and theprotein moiety is an interferon-α moiety, a human growth hormone moiety,or an erythropoietin moiety.
 2. The conjugate of claim 1, wherein theprotein moiety is a modified interferon-α_(2b) moiety containing 1-4additional amino acid residues at the N-terminus.
 3. The conjugate ofclaim 2, wherein the purity of the conjugate is 90% or higher.
 4. Aprotein-polymer conjugate of formula I:

wherein each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C₁₋₅ alkyl,C₂₋₅ alkenyl, C₂₋₅ alkynyl, aryl, heteroaryl, C₃₋₈ cycloalkyl, or C₃₋₈heterocycloalkyl; each of A₁ and A₂, independently, is a polymer moiety;each of G₁, G₂, and G₃, independently, is a bond or a linking functionalgroup; P is a protein moiety; m is 0 or an integer of 1-10; and n is aninteger of 1-10.
 5. The conjugate of claim 4, wherein G₃ is a bond and Pis a protein moiety in which the amino group at the N-terminus is bondedto G₃.
 6. The conjugate of claim 5, wherein each of A₁ and A₂ is an mPEGmoiety having a molecular weight of 10-30 kD.
 7. The conjugate of claim6, wherein each of G₁ and G₂ is

wherein O is attached to A₁ or A₂, and NH is attached to a carbon atomas shown in formula I.
 8. The conjugate of claim 7, wherein P is amodified interferon moiety containing 1-4 additional amino acid residuesat the N-terminus.
 9. The conjugate of claim 8, wherein n is
 2. 10. Theconjugate of claim 9, wherein the conjugate is

in which mPEG has a molecular weight of 20 kD and IFN is aninterferon-α_(2b) moiety.
 11. A compound of formula II

wherein X is a reactive group; each of A₁ and A₂, independently, is apolymer moiety; or one of A₁ and A₂ is a polymer moiety, and the otheris a reactive group; each of G₁, G₂, and G₃, independently, is a bond ora linking functional group; each of R₁, R₂, R₃, R₄, and R₅,independently, is H, C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, aryl,heteroaryl, C₃₋₈ cycloalkyl, C₃₋₈ heterocycloalkyl, or -G₄-X′, G₄ beinga bond or a linking functional group and X′ being a reactive group; andeach m and n, independently, is 0 or an integer of 1-10; provided thatif -G₃-X is —CO(O)—N-succinimidyl, then n is an integer of 1-10.
 12. Thecompound of claim 11, wherein each of A₁ and A₂, independently, is apolymer moiety; each of R₁, R₂, R₃, R₄, and R₅, independently, is H,C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, aryl, heteroaryl, C₃₋₈cycloalkyl, or C₃₋₈ heterocycloalkyl; and X is CH(═O) or a leavinggroup.
 13. The compound of claim 12, wherein X is CH(═O) and G₃ is abond.
 14. The compound of claim 13, wherein each of A₁ and A₂ is an mPEGmoiety having a molecular weight of 12-30 kD.
 15. The compound of claim11, wherein each of G₁ and G₂ is

wherein O is attached to A₁ or A₂, and NH is attached to a carbon atomas shown in formula I; and n is an integer of 1-10.
 16. A method ofpreparing a protein-polymer conjugate of formula I:

wherein each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C₁₋₅ alkyl,C₂₋₅ alkenyl, C₂₋₅ alkynyl, aryl, heteroaryl, C₃₋₈ cycloalkyl, or C₃₋₈heterocycloalkyl; each of A₁ and A₂, independently, is a polymer moiety;each of G₁, G₂, and G₃, independently, is a bond or a linking functionalgroup; P is a protein moiety, in which the amino group at the N-terminusis attached to G₃; m is 0 or an integer of 1-10; and n is an integer of1-10; said method comprising: coupling an N-terminus free protein H—P, Pbeing a protein moiety, with a di-polymer branched molecule of formulaII:

wherein R₁, R₂, R₃, R₄, R₅, A₁, A₂, G₁, G₂, G₃, m and n are as definedabove, and X is a leaving group; or coupling an N-terminus free proteinH—P, P being a protein moiety, with a di-polymer branched molecule offormula II:

wherein R₁, R₂, R₃, R₄, R₅, A₁, A₂, G₁, G₂, and m are as defined above,G₃ is a bond, n is 0 or an integer of 1-9, and X is CHO; and thenreducing the coupling product to form the protein-polymer conjugate. 17.The method of claim 16, wherein each of A₁ and A₂ is an mPEG moietyhaving a molecular weight of 20 kD.
 18. The method of claim 17, whereineach of G₁ and G₂ is

wherein O is attached to A₁ or A₂, and NH is attached to a carbon atomas shown in formula I.
 19. The method of claim 18, wherein P is amodified interferon moiety containing 1-4 additional amino acid residuesat the N-terminus.
 20. The method of claim 19, wherein P is

IFN being an interferon-α_(2b) moiety, the N-terminus of which is bondedto the carbonyl group.
 21. The method of claim 20, wherein G₃ is a bondand X is CH(═O).
 22. A method of treating hepatitis C virus infection orhepatitis B virus infection comprising administering to a subject inneed thereof an effective amount of a protein-polymer conjugate offormula I:

wherein each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C₁₋₅ alkyl,C₂₋₅ alkenyl, C₂₋₅ alkynyl, aryl, heteroaryl, C₃₋₈ cycloalkyl, or C₃₋₈heterocycloalkyl; each of A₁ and A₂, independently, is a polymer moiety;each of G₁, G₂, and G₃, independently, is a bond or a linking functionalgroup; P is an interferon moiety; m is 0 or an integer of 1-10; and n isan integer of 1-10.
 23. A method of treating hepatitis C virus infectionor hepatitis B virus infection comprising administering to a subject inneed thereof an effective amount of a substantially pure conjugatecomprising at least one polymer moiety, a protein moiety, and a linker,wherein the at least one polymer moiety is attached to the linker; thenitrogen atom of the N-terminus of the protein moiety is bonded to thelinker; the linker is a covalent bond, alkylene, alkenyl, or alkynyl;and the protein moiety is an interferon-α_(2b) moiety.